Colistin-resistant Pseudomonas aeruginosa clinical strains with defective biofilm formation

Aim: Colistin is the only effective antibiotic in some cases of Pseudomonas aeruginosa resistance to all tested antibiotics, even carbapenem. On the other hand, biofilm formation is one of the antibiotic resistance mechanisms in this bacterium. The aim of this study was to examine biofilm formation in colistin-resistant P. aeruginosa for the first time. Method: Two groups of P. aeruginosa were included in this study: 1) colistin-resistant and 2) colistin-susceptible. Biofilm formation was determined in these groups using the micro-tube test well as PCR to detect the genes involved in biofilm formation (ppk and molA). The plasmids for colistin resistance, mcr-1 and mcr-2, were also determined. P. aeruginosa ATCC 27853 was used as a control for all tests. Results: Strong biofilm formation was observed only in colistin-susceptible strains, and ppk and modA were not detected in colistin-resistant strains. The control strain P. aeruginosa ATCC 27853 possesses ppk and modA and is categorized as a strong biofilm formation group. According to the results of this study, colistin resistance is associated with defective biofilm formation, as reported by other studies on Acinetobacter baumannii.


Introduction
Multi-drug resistant (MDR) Pseudomonas aeruginosa strains are increasingly isolated from clinical specimens worldwide, which is cause for global concern [1], [2], [3], [4]. In some cases, only colistin remains as an effective antibiotic. However, this is problematic due to the nephrotoxicity of colistin and resistance to it, which can increase morbidity and mortality especially in immunosuppressed patients (e.g, hospitalized persons) [1], [2], [3], [4], [5], [6]. Colistin-resistant P. aeruginosa can be related to two chromosomal mutations: 1) modification of lipid A and 2) loss of LPS [7]. Dafopoulou et al. showed a loss of 47,969 bp genomic regions containing some genes like ppk and modA, which have been previously related to biofilm production in Enterobacteriaceae and Pseudomonas by whole genome mapping [7]. Thus, resistance to colistin can be associated with defective biofilm formation in Pseudomonas. Biofilm formation is one of the antibiotic-resistance mechanisms in P. aeruginosa, and can lead to crossresistance based on the low penetration of antibiotics into the bacterial community after biofilm formation, and the appearance of MDR strains [2], [8]. Alarmingly, in 2016, first reports of plasmid-borne colistin resistance associated with mcr-1 and mcr-2 genes were published [9], [10]. The appearance of plasmid-borne colistin resistance is very important because of horizontal transmission in the bacterial population [9], [10]. The aim of this study was twofold: 1. to examine the relationship between colistin resistance and deficient biofilm formation in clinical P. aeruginosa strains, and 2. to test for mcr-1 and mcr-2 genes in colistin-resistant strains.

Bacterial isolates
Two groups of colistin-resistant and colistin-susceptible strains of P. aeruginosa included 25 strains in each group. These were collected from blood culture and burn wound infections in pediatric and adult patients from two teaching hospitals, in Tehran. Collected strains were identified by conventional biochemical and microbiological tests, e.g. oxidase, TSI, lysine decarboxylase. Resistance to colistin was determined by the disc-diffusion agar method according to CLSI 2015 [11].

Phenotypic biofilm formation detection by micro-tube method
The micro-tube method, as described by Hassan et al., is a qualitative method for biofilm detection. A loop full of test organisms was inoculated in 1 mL of trypticase soy broth with 1% glucose in test tubes. The micro-tubes were incubated at 37°C for 24 h. After incubation, the micro-tubes were decanted and washed with phosphate buffer saline (pH 7.3) and dried. The micro-tubes were then stained with crystal violet (0.1%), and excess stain was rinsed off with deionized water. Micro-tubes were dried in the inverted position. The micro-tube biofilm results were classified as one of three types relative to the results of the control strains: 1) strong/high biofilm formation, 2) moderate, and 3) weak according to the mass of visible film lining the wall of the micro-tubes [12].

Molecular detection of ppk and modA genes
ppk and modA genes were amplified by PCR and specific primers (Table 1) in both colistin-resistant and colistinsusceptible strains. The conditions of PCR have been described previously [2]. DNA was extracted using the boiling method. Positive detection was confirmed by Sanger sequencing. Isolates which showed a specific band after PCR and electrophoresis were sent for sequencing to Pishgam, Macrogen, Seoul, Korea. P. aeruginosa ATCC 27853 was used as reference strain.
Detection of mcr-1 and mcr-2 mcr-1 and mcr-2 were detected in colistin-resistant strains by PCR and specific primers ( Table 1). The PCR product load was determined on 1% agarose and visualized by gel document.

Results
In this cross-sectional study, 25 colistin-resistant and 25 colistin-susceptible isolates were collected. All colistinresistant P. aeruginosa strains were isolated from blood culture, and the colistin-susceptible strains were collected from burn wound infections. Ciprofloxacin and gentamicin are the most effective antibiotics against colistin-resistant and colistin-susceptible strains, respectively (Table 2).

Micro-tube test
Biofilm formation was classified into three groups according to tube test results: A weak, B moderate, and C strong. Strong biofilm formation was only observed in the colistinsusceptible strains (Table 3). These three groups observed in the micro-tubes are depicted in Figure 1. P. aeruginosa ATCC 27853 possesses ppk and modA, and strong biofilm formation was observed.

modA and ppk detection
According to PCR and sequencing results, modA was detected in 23 (92%) of 25 and 17 (68%) of 25 colistinsusceptible strains (Figure 2, Figure 3). But none of the colistin-resistant strains carried these two genes responsible for biofilm formation. The results of sequencing were confirmed by the positive results of the PCR assay.

Mcr-1 and mcr-2 detection
These two plasmid-borne colistin-resistant genes were not detected in any of strains.

Discussion
P. aeruginosa is one of a considerable number of Gramnegative bacteria that can cause nosocomial infections, especially in burn patients [1], [2], [3], [4], [5]. Monitoring P. aeruginosa in health care centers is very important, because it has great ability to survive in the hospital environment, its intrinsic antibiotic resistance mechanisms, and its potential for acquiring antibiotic-resistance genes [1], [2], [4]. Extensive drug-resistant (XDR) and MDR strains of P. aeruginosa can increase morbidity and mortality especially in hospitalized and immune-suppressed patients [1], [2], [3], [4], [5].  Carbapenems are the last line of defence in antibiotic therapy of ESBL-producing P. aeruginosa strains. Unfortunately, carbapenem-resistant strains have been reported worldwide, and in such cases, colistin remains the only effective antibiotic despite its nephrotoxicity [1], [2], [3], [4], [5], [6]. Up to 2016, all of the detected colistinresistance mechanisms were attributed to chromosomal genes. Then, however, a plasmid-borne colistin-resistance gene was reported for the first time from China in 2016. mcr-1 and mcr-2 are both located on the plasmid and can perform horizontal transfer in bacteria [9], [10]. On the other hand, biofilm formation is one of the antibioticresistance mechanisms in P. aeruginosa [2], [8]. This Gram-negative bacterium can produce biofilm on the hospital surfaces and catheters, cause therapeutic complications and consequently prolongation of hospital stays, which finally impose increased health care costs for society and patients [8], [13], [14]. The results of a study in France in 2015 on A. baumannii indicated that chromosomal resistance to colistin is associated with the loss of 47,969 bp genomic regions containing modA and ppk (genes involved in biofilm formation in Pseudomonas), according to whole genome sequencing results [7]. Thus, colistin-resistant A. baumannii strains can exhibit defective biofilm formation [7]. The results of the current study confirmed this observation. The results of the microtube test indicated that biofilm formation was weak and moderate in 84% and 16% of colistin-resistant strains, respectively. In contrast, biofilm formation was strong and moderate in 52% and 32% of colistin-susceptible P. aeruginosa. modA and ppk detection confirmed this hypothesis, because none of the colistin-resistant strains carried both of these genes. On the other hand, all colistin-susceptible strains harbored at least one of these two biofilm formation genes, except two isolates.

Conclusion
According to the results of this study, colistin resistance can be accompanied by defective biofilm formation, similar to the results of Defopoulou et al. on A. baumannii. Future studies on different antibiotic resistances and their combined effect will be very important and helpful in promoting more successful antibiotic therapy and thus better health, especially pediatric patients.

Notes Funding
The research reported in this publication was supported by Elite Researcher Grant Committee under award number [958699] from the National Institutes for Medical Research Development (NIMAD), Tehran, Iran.